Compounds suited as nanocarriers for active agents and their use

ABSTRACT

The invention relates to a compound suited as entity carrier, having the general formula (I) 
     
       
         
         
             
             
         
       
     
     wherein X is an amine-containing residue further defined herein. The invention further relates to the use of such compounds, a nanocarrier system, a kit comprising such compounds and methods for gene silencing and anti-cancer treatment.

The invention relates to a nanocarrier system according to the preambleof claim 1, a compound being suited as entity carrier according to thepreamble of claim 4, the uses of such a compound according to thepreamble of claims 9 and 10, a kit comprising such a compound accordingto the preamble of claim 14 and application methods according to claims15 and 17.

Gene therapy provides great opportunities for treating diseases likegenetic disorders, infections, and cancer (T. G. Park et al. AdvancedDrug Delivery Reviews, 2006, 58, 467-486). Double stranded RNA (dsRNA)induces sequence-specific post-transcriptional gene silencing by aprocess known as RNA interference (RNAi). The mediators of RNAi aresmall interference RNA (siRNA) segments of 21 to 25 base pairs inlength.

These siRNAs bind to a ribonuclease complex called RNA-induced silencingcomplex (RISC) that guides the siRNA to its homologous mRNA targets. Asa result, the bounded mRNA is cleaved; degradation of the mRNA resultsin gene silencing.

To achieve successful gene therapy, development of proper gene deliverysystems is the main obstacle. For the uptake of DNA/siRNA varioussystemic and cellular barriers have to be circumvented.

A large variety of cationic compounds were reported to efficientlydeliver nucleic acids or other biomolecules or even other substancessuch as metal ions into the cell. Generally, cationic compounds areneeded to carry nucleic acids into a cell since the latter show anoverall negative charge (due to their phosphate backbone) so that acharge interaction between the carrier and the nucleic acid to becarried can occur.

One of the most powerful and versatile families of carriers arepolyamines. However, these polyamines exert rather high cell toxicityand low biocompatibility. Therefore, polyamines are not well suited ascarriers for in vivo applications.

Cationic lipids, such as the HiPerFect reagent of Qiagen, Hilden,Germany, are also used as carrier compounds. In this context, HiPerFectis the benchmark reagent for in vitro transfections. However, due to itscell toxicity, it is not well suited as siRNA carrier for in vivoapplications.

Other siRNA carriers include the RNAiFect reagent (Qiagen), DOTAP(Roche), lipofectamine (Gibco) and polyethylene imine. All compoundsshow also significant cell toxicity and are thus only suited for invitro applications.

Roller et al. (S. Roller, H. Zhou, R. Haag, Molecular Diversity, 2005,9, 305-316) describe different amine-substituted polyglycerol-basedpolymeric scaffolds. However, no use of these compounds as carriers hasbeen proposed hitherto. In addition, N-Benzyl-O-polyglyceryl carbamatedescribed by Roller et al. is not at all suited as carrier since it istoo hydrophobic and is not water soluble.

Tziveleka et al. (L.-A. Tziveleka, A.-M. G. Psarra, D. Tsiourvas, C. M.Paleos, International Journal of Pharamceutics, 2008, 356, 314-324)describe five derivatives of hyperbranched polyether polyols beingfunctonalized with quarternary or tertiary ammonium groups. Thesederivatives may be used to carry plasmidic DNA (pDNA).

For a transfection to be therapeutically successful, it is imperativethat polymeric scaffolds to be used as carriers exert reduced celltoxicity and higher biocompatibility.

It is an objective of the invention to provide novel compounds beingsuited as carriers for diverse substances, also in vivo, as well as toprovide a nanocarrier system and to provide a novel use of already knowncompounds.

This objective is attained by a nanocarrier system according to claim 1.Such a nanocarrier system comprises at least one nanocarrier being acompound having a structure according to formula (I),

wherein PG denotes a linear or branched polyglycerol core, and

-   X being

preferably

-    and being covalently bound to a carbon atom of the polyglycerol    core, wherein the polyglycerol core carries a plurality of groups of    the type X,-   R¹ being H, linear or branched C₁-C₁₀-Alkyl, which may be    substituted and/or interrupted by one or more oxygen, sulphur and/or    nitrogen atoms, or R³,-   R² being H, linear or branched C₁-C₁₀-Alkyl, which may be    substituted and/or interrupted by one or more oxygen, sulphur and/or    nitrogen atoms, or R³,-   R³ being

-   R⁴ being H or C₁-C₄-Alkyl, which may be substituted and/or    interrupted by one or more oxygen, sulphur and/or nitrogen atoms,    and-   n being 1 to 100.

The nanocarrier system further comprises at least one entity to becarried by and bound to said nanocarrier in a covalent, ionic orcomplexed manner, wherein said entity is chosen from the groupcomprising nucleotides, nucleosides, linear or circular single or doublestranded oligonucleotides, oligomeric molecules comprising at least onenucleoside, small pharmacologically active molecules having a molecularmass of not more than 800 g/mol, amino acids, peptides, and metal ions.

In this context, the entity of the claimed nanocarrier system doespreferably not comprise double stranded circular and covalently closednucleic acid species (preferably DNA or RNA, in particular DNA) having alength of more than 1000 bases or base pairs, preferably of more than500 bases or base pairs. Thus, plasmidic DNA, i.e. double strandedcircular and covalently closed DNA having more than 1000 base pairs ormore than 500 base pairs preferably cannot be part of the nanocarriersystem. However, in certain embodiments, it is possible that the entitymay also be plasmidic DNA according to the definition given above.

In an embodiment, the entity of the nanocarrier system is a ribonucleicoligonucleotide, i.e. an RNA oligonucleotide. In another embodiment, theribonucleic oligonucleotide is a messenger RNA (mRNA) or an mRNAanalogue, a micro RNA (miRNA), a small interfering RNA (siRNA) or a tinynoncoding RNA (tnRNA).

In another embodiment, the oligonucleotide is a double stranded circularcovalently closed deoxyribonucleic or ribonucleic oligonucleotide with alength of ca. 20 to less than 1000 base pairs, preferably of ca. 20 toless than 500 base pairs.

In another embodiment, the ribonucleic oligonucleotide has a length of 8to 50 bases, preferably 10 to 40 bases, preferably 10 to 30 bases,preferably 12 to 25 bases, preferably 12 to 22 bases.

In any case, the bases which might be part of the nucleotides,nucleosides, or oligonucleotides used as or in the entity of thenanocarrier system, can be natural or non-natural bases. E.g., suitednucleoside analogues include, but are not limited to inosine,nebularine, nitropyrrole, nitroindole, 2-aminopurine, 2,6-diaminopurine,locked nucleic acids (LNAs) nucleosides, peptide nucleic acids (PNAs)nucleosides, purine, hypoxanthine, xanthine, ethanocytosin,5-methylcytosine, 5-alkynylcytosine, 2,6-diaminopyrimidino,2,6-diaminopyrazine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin,isocytosine, isoguanine, inosine, 4-acetylcytosine, dihydrouracil,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, uracil-5-oxyacetic acid methylester,5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl), pseudouridine,pseudocytosine, pseudoisocytosine, 5-propynylcytosine, isocytosine,isoguanine, 2-thiopyrimidine, 6-thioguanine, 4-thiothymine,4-thiouracil, O⁶-methylguanine, N⁶-methyladenine, O⁴-methylthymine,5,6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, andethenoadenine.

Accordingly, the nucleotides can be modified in this manner. Theoligonucleotides may be LNAs or PNAs. PNA is a polymer of purine andpyrimidine bases which are connected to each other via a 2-amino ethylbridge. PNA binds sequence specifically with high affinity to anaccording DNA or RNA complement. In LNA, the 2′-hydroxyloxygen of riboseis connected to the C-4 atom of the same ribose unit via a methylenebridge. The conformational restriction of LNA compared to RNA or DNAapparently leads to a higher binding affinity.

All possible meanings for the entity as defined above are to beunderstood as individually disclosed herein and to be optionallycombined in any desired manner.

In an embodiment, the nanocarrier system comprises a polyglycerol corein which at least 50%, particularly at least 60%, particularly at least70%, particularly at least 80%, particularly at least 90%, particularlyat least 95%, particularly at least 99%, particularly all of the freehydroxyl groups of the polyglycerol core are substituted by groups ofthe type X.

In another embodiment, n is preferably 1 to 10, particularly 5.

In another embodiment, the nanocarrier system preferably comprises acompound in which R¹ is a methyl residue and R² is an N-dimethyl ethylamine residue.

The objective is also attained by a compound having the features ofclaim 1. Such a compound is suited as entity carrier and has the generalformula (I)

with

-   PG denoting a linear or branched polyglycerol core,-   X being

and being covalently bound to a carbon atom of the polyglycerol core,wherein the polyglycerol core carries a plurality of groups of the typeX,

-   R¹ being H, linear or branched C₁-C₁₀-Alkyl, which may be    substituted and/or interrupted by one or more oxygen, sulphur and/or    nitrogen atoms, or R³,-   R^(2′) being linear or branched C₁-C₁₀-Alkyl, which may be    substituted and/or interrupted by one or more oxygen, sulphur and/or    nitrogen atoms, or R³,-   R³ being

-   R⁴ being H or C₁-C₄-Alkyl, which may be substituted and/or    interrupted by one or more oxygen, sulphur and/or nitrogen atoms,    and-   n being 1 to 100,    wherein R¹ and R^(2′) cannot simultaneously be an ethyl residue.

In an embodiment, the entity to be carried by said compound suited asentity carrier is chosen from the group comprising nucleotides,nucleosides, linear or circular single or double strandedoligonucleotides, oligomeric molecules comprising at least onenucleoside, small pharmacologically active molecules having a molecularmass of not more than 800 g/mol, amino acids, peptides, and metal ions.In an embodiment, the afore-mentioned group does not comprise DNA orplasmidic DNA or essentially completely double stranded nucleic acids orany combination thereof.

E.g., a single nucleoside (of ribonucleic acid or of deoxyribonucleicacid) like uridine or deoxythymidine or a plurality of identical ordifferent nucleosides may serve as entity. Examples of nucleotides areadenosine monophosphate (AMP), adenosine diphosphate (ADP) and adenosinetriphosphate (ATP). Also, different metal ions, particularly cationssuch as Ag⁺, Ca²⁺, Cu²⁺ or Mg²⁺ ions, or aptamers of peptides oroligonucleotides may serve as entity. In case of the entity being metalions, preferably a non-covalent, complexed form of interaction betweenthe metal ions and the carrier is established.

Another example of a suited entity are single or double stranded RNA orDNA oligomers of, e.g., 20 to 25 base pairs or bases, respectively, inlength. Double stranded RNA is particularly suited. Thus, the compoundmay also serve as siRNA carrier. Another example of suited entities arechimeric molecules of amino acid residues and nucleosides ornucleotides.

Reference is also made to the explanations given above with respect tothe nanocarrier system which are also applicable for the compound andits use.

All possible meanings for the entity as defined above are to beunderstood as individually disclosed herein and to be optionallycombined in any desired manner.

As can be seen from formula (I) and the residue definitions given above,the claimed compound has a polyglycerol (PG) based gene-transfectionmotif with core-shell architecture. The shells of such motifs can betailored to contain amines with different numbers of cationic sites formimicking the activity of polyamines. Since the compounds are based on aPG structure, they provide appreciable clinical compliance.

In contrast to polyamines and other known compounds used as carriers,the novel compounds carry charges at physiological pH only on theirsurface or shell (namely on nitrogen atoms located on the surface orbeing part of the shell), whereas the core is comprised of short alkylchains connected via ether bridges to each other being substantially notcharged. The polyglycerol core may be structured in a linear or branchedmanner. In an embodiment, the structure of the polyglycerol is at leastpartially branched.

The shell of the polyglycerol-based compounds may have a layeredstructure due to a repetitive nitrogen-containing motif. E.g., by use ofa pentaethylenehexamine residue as shell (as is the case in polyglycerylpentaethylenehexamine carbamate), a five-fold layered shell is achieved.

The polyglycerol base material can be obtained in a kilogram scale whichcontains linear monohydroxy and terminal dihydroxy functionalities whichcan be modified selectively as linkers for diverse organic synthesis.

The polyglycerol core of the claimed compounds is biocompatible.However, by introducing nitrogen-containing shell motifs, the celltoxicity of the compounds is raised. Thus, when designing carriers to beespecially used for in vivo applications, care must be taken to keep thecell toxicity of the complete compound at a low level. On the otherhand, the transfection efficacy should be as high as possible.Consequently, a balance must be found between toxicity and transfectionefficacy. Regarding the claimed compounds, such a balance isestablished.

Specific, symmetric polyglycerol dendrimers are an example ofpolyglycerol which can be used for the polyglycerol core of thenanocarrier system or the compound. These dendrimers are highlysymmetric. They are generated from smaller molecules by repeatedreaction steps, wherein always higher degrees of branching result. Atthe end of the branches, functional groups are located which are thestarting point for further branchings. Thus, with each reaction step,the number of monomeric end groups increases exponentially, leading to ahemicircular tree structure.

In this context, the term “polyglycerol” as used herein includes anysubstance which contains at least two glycerol units in its molecule andwherein said molecule is characterized by a branched structure.According to the present invention, the term “glycerol unit” does notonly relate to glycerol itself but also includes any subunits which arebased on glycerol, such as for example:

Preferably, the polyglycerol includes three or more, preferably ten ormore, and particularly 15 or more of said glycerol units. Thepolyglycerol structure can be obtained, e.g., by a perfect dendrimersynthesis, a hyperbranched polymer synthesis or a combination of bothand is per se known to a person skilled in the art.

In an alternative embodiment, at least 50%, particularly at least 60%,particularly at least 70%, particularly at least 80%, particularly atleast 90%, particularly at least 95%, particularly at least 99%,particularly all of the free hydroxyl groups of the polyglycerol core ofthe compound are substituted by groups of the type X. The rate ofsubstitution is also referred to as conversion. Thus, if a conversion of100% is achieved during synthesis, the starting material polyglycerol ofthe formula PG-(OH)_(p) was reacted to PG-(X)_(m) with m=p. If, e.g., aproduct of the formula (X)_(m)—PG-(OH)_(q) with m=0.8*n and q=0.2*p isobtained, the conversion is 80%.

As an alternative, n is 1 to 10, particularly 2 to 8, particularly 3 to6 and in particular 5. As another alternative, R⁴ is H. If X is

n is 5 and R⁴ is H, the compound would be polyglycerylpentaethylenehexamine carbamate.

In another embodiment, R¹ is a methyl residue and R^(2′) is anN-dimethyl ethyl amine residue so that anN,N,N′-trimethylethylenediamine residue is bound to the polyglycerolcore structure via one of its nitrogen atoms.

The objective is also achieved by a use of a compound as defined above(with all alternative embodiments) with respect to the claimed compoundor of a compound according to general formula (I), wherein X is

according to claim 9. This use is directed to the preparation of apharmaceutical composition, wherein the compound acts as carrier for anentity, wherein the entity is chosen from the group comprisingnucleotides, nucleosides, linear or circular single or double strandedoligonucleotides, oligomeric molecules comprising at least onenucleoside, small pharmacologically active molecules having a molecularmass of not more than 800 g/mol, amino acids, peptides, and metal ions.However, it is mandatory that the entity does not comprise a doublestranded circular and covalently closed nucleic acid (preferably DNA orRNA, in particular DNA) having a length of more than 1000 bases or basepairs, preferably of more than 500 bases or base pairs.

Examples of entities are disclosed above. All possible meanings for theentity as defined above are also in the context of the claimed uses tobe understood as individually disclosed herein and to be optionallycombined in any desired manner.

The pharmaceutical composition may be used to treat diverse diseases,such as diseases which are amenable to treating by gene silencing like,e.g., certain types of cancer. Further, in particular when Cu²⁺ ions areused as entity, the pharmaceutical composition may serve for slowingdown aging.

The objective is also achieved by the use of a compound of the generalformula (I) as explained above (with all alternative embodiments) withrespect to the claimed compound or of a compound according to generalformula (I), wherein X is

as entity carrier for in vitro, in vivo, ex vivo or in situapplications.

In this context, the entity is chosen from the group comprisingnucleotides, nucleosides, linear or circular single or double strandedoligonucleotides, oligomeric molecules comprising at least onenucleoside, small pharmacologically active molecules having a molecularmass of not more than 800 g/mol, amino acids, peptides, and metal ions.However, at least in case of R¹ and R² both being an ethyl residue, theentity does not comprise a double stranded circular and covalentlyclosed nucleic acid (preferably DNA or RNA, in particular DNA) having alength of more than 1000 bases or base pairs, preferably of more than500 bases or base pairs. If the polyglycerol core is substituted by acarbamate residue or a residue of the general structure

plasmidic nucleic acids (i.e. double stranded circular and covalentlyclosed nucleic acids) are also suited as entity in an embodiment.

In an alternative embodiment, the use is directed only to in vitro, exvivo or in situ applications, but not to in vivo applications. Inanother alternative embodiment, the use is directed only to in vitro orex vivo applications, but not to in vivo or in situ applications.

In another embodiment, it is preferred to use the entity carrier for invivo or in situ applications.

An example of such a use or a use as pharmaceutical composition is toutilize the entity carrier to transport said entity into at least oneprokaryotic or eukaryotic cell, in particular into at least one human oranimal cell. Transporting said entity in a plurality of entity carriersinto a plurality of cells is preferred. Suited animal cells are, e.g.,cells of mammals like, e.g., rodents such as rats or mice.

In an alternative embodiment, the entity carrier is used to transportentity into at least one animal cell but not into a human cell. Thus,the use of the compound as entity carrier may be defined as for invitro, in vivo, ex vivo or in situ applications with respect to animalcells and for in vitro, ex vivo or in situ applications for human cells.

In an embodiment, the use is directed to silence a gene within a cell,preferably a tumor gene, by a ribonucleic acid, such as siRNA.

In another embodiment, the entity carrier bears at least one functionalgroup of the general formula

wherein residues R³ and R⁴ have the above-defined meanings, and in thatsaid functional group is cleaved from the polyglycerol core of theentity carrier once the entity carrier is located within its targetcell. This cleavage results in an even better biocompatibility of thecompound, since potentially cytotoxic amine structures of the compoundlike polyamine or polyethyleneamine structures are separated from thegenerally biocompatible polyglycerol core structure.

In another embodiment, said cleavage is performed by an enzyme. E.g.,the compound may be designed in such a way that an esterase or acarbamate hydrolase may cleave the carbamate bond so that thepolyglyceryl core is separated from the surrounding amine-containingsurface or shell.

Again, all possible meanings for the entity as defined above are also inthe context of the claimed nanocarrier system to be understood asindividually disclosed herein and to be optionally combined in anydesired manner.

The invention also relates to a kit for performing transfectionreactions, comprising a compound of general formula (I) as defined abovein any suited formulation. E.g., a formulation as a solution in aphosphate buffered saline (PBS) at pH 7.4 or as a lyophilized productwith or without adducts may be suited. Other buffer systems can also beused.

The object is also achieved by a method for silencing a gene in vitro,ex vivo, in situ or in vivo according to claim 15. This method ischaracterized by applying a nanocarrier system as described above or acompound as described above together with at least one entity to becarried by and bound to the mentioned compound in a covalent, ionic orcomplexed manner into at least one human or animal cell. Thereby, theentity is a ribonucleic oligonucleotide.

In an alternative embodiment, the method is directed only to in vitro orex vivo applications, but not to in vivo or in situ applications. Inanother alternative embodiment, the method is directed only to in vitro,in situ or ex vivo applications, but not to in vivo applications.

The application of the nanocarrier system or the compound/ribonucleicacid is done such that the ribonucleic acid can interact with mRNA beingpresent in said cell.

In another embodiment, it is preferred to use the method for in vivo orin situ applications.

In an embodiment, the gene to be silenced is a tumor related gene.

The object is further achieved by a method for the treatment of canceraccording to claim 17. This method is characterized by administering ananocarrier system as described above or a compound as described abovetogether with at least one entity to be carried by and bound to saidcompound in a covalent, ionic or complexed manner, to at least one humanor animal being. Thereby, the entity is chosen from the group comprisingnucleotides, nucleosides, linear or circular single or double strandedoligonucleotides, oligomeric molecules comprising at least onenucleoside, small pharmacologically active molecules having a molecularmass of not more than 800 g/mol, amino acids, peptides, and metal ions

In an embodiment, the treatment of cancer is performed as combinationtreatment by a combined administering of the nanocarrier system or thecompound/entity according to the invention together with a knownanti-cancer or an anti-angiogenic drug. Exemplary cytotoxic agentssuited as anti-cancer drug include, without limitation, anthracyclineantibiotics like doxorubicin and daunorubicin; taxanes like paclitaxelTaxol™, docetaxel; vinca alkaloids like vincristine and vinblastine,anti-metabolites like methotrexate, 5-fluorouracil (5 FU), leucovorin,irinotecan, idarubicin, mitomycin C, oxaliplatin, raltitrexed, tamoxifenand cisplatin, carboplatin, actinomycin D, mitoxantrone or blenoxane ormithramycin.

Exemplary anti-angiogenic drugs include, but are not limited to: (1)monoclonal antibodies directed against specific proangiogenic factorsand/or their receptors; (avastin, erbitux, vectibix, herceptin) and (2)small molecule tyrosine kinase inhibitors (TKIs) of multipleproangiogenic growth factor receptors (tarceva, nexavar, sutent,iressa). Inhibitors of mTOR (mammalian target of rapamycin) represent athird, smaller category of antiangiogenic therapies with one currentlyapproved agent (torisel). In addition, at least two other approvedanti-angiogenic agents may indirectly inhibit angiogenesis throughmechanisms that are not completely understood (velcade,thalidomide/celgene). Other anti-angiogenic agents that are suitable foruse in the context of embodiments of the invention include, but are notlimited to, paclitaxel, 2-methoxyestradiol, prinomastat, batimastat, BAY12-9566, carboxyamidotriazole, CC-1088, dextromethorphan acetic acid,dimethylxanthenone acetic acid, endostatin, IM-862, marimastat, a matrixmetalloproteinase, penicillamine, PTK787/ZK 222584, RPI.4610, squalaminelactate, SU5416, thalidomide, combretastatin, tamoxifen, COL-3,neovastat, BMS-275291, SU6668, anti-VEGF antibody, medi-522 (vitaxinII), CAI, interleukin-12, IM862, amilloride, Angiostatin®protein,angiostatin K1-3, angiostatin K1-5, captopril,DL-alpha-difluoromethylornithine, DL-alpha-difluoromethylornithine HCl,His-Tag® Endostatin™ protein, Endostar™, fumagillin, herbimycin A,4-Hydroxyphenylretinamide, juglone, laminin, laminin hexapeptide,laminin pentapeptide, lavendustin A, medroxyprogesterone,medroxyprogesterone acetate, minocycline, minocycline HCl, placentalribonuclease inhibitor, suramin, sodium salt suramin, human plateletthrombospondin, neutrophil granulocyte; interferon alpha, beta andgamma; IL-12; matrix metalloproteinases (MMP) inhibitors (e.g. COL3,Marimastat, batimastat); EMD121974 (cilengitide); ZD6474, SU11248,Vitaxin; squalamin; COX-2 inhibitors; PDGFR inhibitors (e.g., gleevec);NM3 and 2-ME2.

Alternative embodiments of the nanocarrier system, the compound or theentity as indicated above are also independently applicable for theclaimed methods.

The invention will be explained in the following with reference tofigures and examples. This will be done for better understanding of theinvention is not intended to limit the scope of protection in any way.

In the Figures:

FIG. 1 shows a cut-out of the polyglycerol core of a compound accordingto general formula (I),

FIG. 2 shows a reaction scheme for the synthesis of differentpolyglyceryl ethyleneamine carbamates as examples of a compoundaccording to formula (I),

FIG. 3 shows a reaction scheme for the synthesis of different aminoanalogues of polyglycerol as further examples of compounds according toformula (I),

FIG. 4 shows in vitro siRNA gene silencing efficiency of siRNA loadedPG-(C₂H₄NH)₅ (WF33) and PG-NH₂ (SX118) with respect to Lamin expressionin HeLaS3 cells,

FIG. 5 shows in vitro siRNA gene silencing efficiency of siRNA loadedPG-(C₂H₄NH)₅ (WF33) and PG-NH₂ (SX118) with respect to CDC2 expressionin HeLaS3 cells,

FIG. 6 shows in vitro siRNA gene silencing efficiency of siRNA loadedPG-(C₂H₄NH)₅ (WF33) and PG-NH₂ (SX118) with respect to MAPK2 expressionin HeLaS3 cells,

FIG. 7 shows in vitro siRNA gene silencing efficiency of siRNA loadedPG-(C₂H₄NH)₅ and PG-NH₂ with respect to luciferase expression in U87-Luccells,

FIG. 8 shows in vitro cytotoxicity of PG-(C₂H₄NH)₅ (WF33) and PG-NH₂(SX118) on HeLaS3 cells,

FIG. 9 shows in vitro cytotoxicity of PG-(C₂H₄NH)₅ (WF33) and PG-NH₂(SX118) on HeLaS3 cells,

FIG. 10A shows the cytotoxicity of PG-NH₂ and PG-(C₂H₄NH)₅ on SK—N—SHneuroblastoma cells,

FIG. 10B shows the cytotoxicity of PG-NH₂ and PG-(C₂H₄NH)₅ on Kellyneuroblastoma cells,

FIG. 10C shows the cytotoxicity of PG-NH₂ and PG-(C₂H₄NH)₅ on U87 humanglioblastoma cells,

FIG. 10D shows the results of a red blood cell lysis assay using PG-NH₂and PG-(C₂H₄NH)₅, compared to positive (triton X and PEI) and negative(dextran) controls,

FIG. 11A shows luciferase activities within generated tumors of mice atdifferent measurement times as bioluminescence images,

FIG. 11B shows representative bioluminescence images of a SCID mousebearing a human U87-Luc glioblastoma tumor inoculated subcutaneously,that was treated intratumorally with low dose of luciferasesiRNA-PG-NH₂, at different times,

FIG. 11C shows the same representation as FIG. 11B, but in colour,

FIG. 12A shows the luciferase activities of FIG. 11A as percent of theinitial luciferase activity,

FIG. 12B shows the luciferase silencing activity normalized to tumorvolume plotted versus time of SCID mouse bearing a U87-Luc tumor treatedintratumorally with luciferase siRNA-PG-NH₂ or luciferasesiRNA-PG-(C₂H₄NH)₅,

FIG. 13A shows body weight change of animals after treatment withdifferent doses of SX 118,

FIG. 13B shows body weight change of animals after intratumoraltreatment with different doses of siRNA-PG-NH₂ or siRNA-PG-(C₂H₄NH)₅,

FIG. 13C shows the results of a biocompatibility evaluation ofintravenous administration of PG-NH₂ at two different concentrationsinto mice, presented as change in body weight following treatment vs.time,

FIG. 14 shows the hydrodynamic diameter size distribution of PG-NH₂ andPG-(C₂H₄NH)₅,

FIG. 15 shows the results of a gel mobility-shift assay of siRNAincubated with PG-NH₂, or PG-(C₂H₄NH)₅, PEI-PAMAM (i.e., poly(ethyleneimine)-polyamidoamine) or PEI-Gluconolacton (i.e. poly(ethylene imine)gluconolacton) at several molar ratios,

FIG. 16A shows confocal microscopy images (XY image plane) of fixed U87cells transfected with Cy3-labeled anti-luciferase siRNA complexed withPG-NH₂,

FIG. 16B shows the same representation as FIG. 16A, but in colour,

FIG. 16C also shows confocal microscopy images (XZ image plane) of fixedU87 cells transfected with Cy3-labeled anti-luciferase siRNA complexedwith PG-NH₂,

FIG. 16D shows the same representation as FIG. 16C, but in colour,

FIG. 16E shows brightfield and fluorescence images of live cellsprepared in suspension as results obtained by an ImageStreammultispectral imaging flow cytometer of U87 cells transfected withFITC-labeled anti-Luciferase siRNA complexed with PG-NH₂,

FIG. 16F shows intracellular internalization histograms as analyzed flowcytometric results of the experiment of FIG. 16E,

FIG. 16G shows levels of siRNA accumulated in the cells cytoplasm (meanfluorescence) as further flow cytometric results of the experiment ofFIG. 16E,

FIG. 17 shows the chemical structures of different natural polyamines,

FIG. 18A shows two reaction schemes for the synthesis of nanocarriers byglycerol oxidation and amination and

FIG. 18B shows a reaction scheme for the synthesis of nanocarriers by aMitsunobu reaction.

FIG. 1 shows a cut-out of the polyglycerol core of a compound accordingto general formula (I). The grade of branching of the polyglycerolstructure can differ from that depicted in FIG. 1. Also, the molecularmass of a polyglycerol core structure of the claimed invention can beequal to that shown in FIG. 1 or can be lower or higher. Duringsynthesis of the compounds according to general formula (I),substitution reactions take place at free hydroxyl residues (—OH) of thepolyglycerol structure. In the abbreviated structure

only a single hydroxyl group is indicated. However, this single hydroxylgroup is to be understood as representative of all free hydroxyl groupsbeing present in the polyglycerol.

FIGS. 2 and 3 showing reaction schemes will be explained in conjunctionwith examples 1 to 3.

FIGS. 4 to 7 showing siRNA silencing efficiencies of two siRNA loadednanocarriers with respect to different protein expressions will beexplained in the context of example 4.

FIGS. 8, 9 and 10A to 10D relating to cytotoxicity experiments will beexplained in conjunction with example 5.

FIGS. 11 and 12 relating to an in vivo gene silencing experiment will beexplained in conjunction with example 6.

FIG. 13 relating to body weight change of animals after treatment with ananocarrier will be explained in conjunction with example 7.

FIG. 14 will be explained in conjunction with Example 8.

FIG. 15 will be explained in conjunction with Example 9.

FIG. 16 will be explained in conjunction with Example 10.

FIGS. 17 and 18 will be explained in conjunction with Example 11.

EXAMPLE 1 Synthesis of Polyglyceryl Ethyleneamine Carbamates

The synthesis of polyglyceryl ethyleneamine carbamates as examples ofamine terminated polyglycerol (PG) compounds was carried out in atwo-step protocol (see FIG. 2). In the first step, hyperbranchedpolyglycerol 1 (cf. also FIG. 1) was activated to phenyl polyglycerolcarbonate 2. In the second step, this activated polyglycerol was reactedwith amines of different chain length to form amine terminatedpolyglycerols 8. By this reaction pathway, it is possible to synthesizea library of different amine derivatives based on a PG core.

As an example of polyglyceryl ethyleneamine carbamates, the synthesis ofpolyglyceryl pentaethylenehexamine carbamate will be explained in moredetail.

1a) Synthesis of phenyl polyglyceryl carbonate (carbonic acidphenylpolyglyceryl ester) 2

This reaction was performed under an inert gas atmosphere and exclusionof water. To absolute (abs.) pyridine (100 ml) in a three necked 500-mlflask with drop funnel, thermometer, and magnetic stirrer was addedwhile stirring at 0° C. phenyl chloroformate 5 (19.6 ml, 24.4 g, 156mmol, 1.2 equivalents (eq.)). On addition a white precipitate formed.

Subsequently, a solution of polyglycerol 1 (10.0 g, 135 mmol OH-groups)in abs. pyridine (80 ml) was added at 0° C. The mixture was stirred inthe thawing cooling bath for 16 h. Then H₂O and CHCl₃ were added untilall solid was dissolved. The phases were separated and the organic layerwas extracted three times with CHCl₃. The combined organic layers weredried over MgSO₄, concentrated in vacuo and dialysed in CHCl₃ to give abrown honey-like product.

Conversion: quantitative (quant.); yield: 92%; ¹H-NMR (500 MHz, CDCl₃):δ=7.61-6.87 (Ar—H), 5.32-4.92 (functionalised secondary PG-groups),4.76-4.10 (functionalised primary PG-groups), 4.05-3.06 (PG), 1.73(PG-starter), 0.86 (PG-starter); ¹³C-NMR (125 MHz, CDCl₃): δ=153.5(1-C), 151.1 (2-C), 129.6 (4-C/6-C), 126.2 (5-C), 121.1 (3-C/7-C); IR(KBr): v=3125-2750, 1761, 1592, 1494, 1458, 1237, 1072, 1023, 913, 775,727, 688 cm⁻¹.

1b) Synthesis of Polyglyceryl pentaethylenehexamine carbamate 7

A solution of phenyl polyglyceryl carbonate 5 (2.215 g, 11.15 mmol, 1eq.) in p.a. pyridine (80 ml) was dropped over 2 h at 0° C. to anemulsion of pentaethylenehexamine 6 (25.92 g, 111.56 mmol, 10 eq.) and4-(Dimethylamino)-pyridine (DMAP; 0.027 g, 0.2231 mmol, 0.01 eq.) inpyridine. The mixture was refluxed for 16 h and after cooling,concentrated in vacuo. After removal of pyridine residues in highvacuum, the raw product was dialyzed in methanol (MeOH) to give a brownhoney-like product.

Conversion: 67%; yield: 73%; ¹HNMR (250 MHz, CD₃OD): δ=4.40-3.03 (PG),1.41 (PG-starter), 0.89 (PG-starter); ¹³CNMR (75 MHz, D₂O): δ=159.4(CO), 81.2-59.4 (PG); IR (KBr): v=3328, 2876, 2361, 1721, 1630, 1514,1461, 1278, 1180, 1077 cm⁻¹.

¹HNMR (MeOD, 250 MHz): δ (ppm)=2.5-2.9 (m, H1-H9), 3.1-3.2 (H11),3.3-3.9 (PG), 4.2-4.4 (H12).

¹³CNMR (125 MHz, CD₃OD): δ=40.2-50.0 (pentaethylenehexamine), 52.9-80.0(PG-backbone), 158.15 (CO).

EXAMPLE 2 Synthesis of N,N,N′-trimethylethylenediamine terminatedpolyglycerol

The synthesis of N,N,N′-trimethylethylenediamine terminated Polyglycerolas another example of an amine terminated polyglycerol (PG) was alsocarried out in a two step protocol (see FIG. 3). In the first step,hyperbranched polyglycerol 1 (cf. also FIG. 1) was activated topolyglycerol mesylate (mesylpolyglycerol) 3. In the second step, thisactivated polygylcerol was reacted with N,N,N′-trimethylethylenediamineto form N,N,N′-trimethylethylenediamine terminated polyglycerol.

2a) Synthesis of mesylpolyglycerol 3

Polyglycerol 1 (10 g, 135 mmol OH-groups) in a three necked flask with adrop funnel, thermometer, and magnetic stirrer was dissolved in abs.pyridine (80 ml). The solution was cooled down to 0° C. by means ofice/NaCl bath. A solution of mesylchloride (12.5 ml, 18.6 g, 162.16mmol, 1.2 eq.) in abs. pyridine was added in a drop wise pattern. Thereaction mixture was stirred for 16 h in the thawing ice bath. Then icewas added and a dark brown solid precipitated. After decantation ofliquid, the solid was washed with H₂O, dissolved and dialyzed in acetonefor overnight to give a brown honey-like product (mesylpolyglycerol 3)at 85% yield. The product was characterized by ¹H and ¹³C NMR and was incomplete compliance with spectroscopic assignments.

2b) Synthesis of Polyglyceryl trimethylethylenediamine 10

To a solution of mesylpolyglycerol 3 (0.5 g, 1 eq.) in dimethylformamide(DMF), was added N,N,N′-Trimethylethylenediamine 9 (2.1 ml, 5 eq.) in asealed tube. The reaction mixture was stirred for 72 h at 90° C. Afterremoval of DMF by cryo-distillation, the condensed reaction mixture wasdissolved in CHCl₃ and extracted three times with saturated NaHCO₃solution. Organic phases were collected and dried over MgSO₄. Afterfiltration and evaporation of CHCl₃, a brown semisolid paste likeproduct was obtained. Dialysis of the crude product against MeOH for 48h yielded amine terminated polyglycerol 10 at 80% yield. Productcharacterization was done by ¹H and ¹³C NMR. Final product was found tobe soluble in water, MeOH and DMF.

EXAMPLE 3 Synthesis of Polyglycerylamines

Polyglycerylamines were synthesized by mesylation of PG hydroxyl groups,conversion into azides and subsequent reduction to amines (see FIG. 3).

3a) Synthesis of polyglycerylazide 12

In a 500 ml one-necked flask with reflux condenser and magnetic stirrerwas dissolved O-mesylpolyglycerol 3 (14.74 g, 90.96 mmol OMs groups) inpro analysi (p.a.) DMF (150 ml) upon ultrasonification. After additionof NaN₃ (29.57 g, 454.8 mmol, 5 eq.), the resulting suspension washeated at 60° C. for 3 days behind a transparent security wall. Aftercooling, filtration delivered a reddish filtrate and a white residue ofexcess NaN₃. The filtrate was concentrated in vacuo at temperaturesbelow 40° C. and only handled with plastic spatula to avoid thepotentially explosive degradation of the polyazide. The remainder wasdissolved in CHCl₃ and extracted four times with water. The organicphase was dried over MgSO₄ and concentrated in vacuo. To remove tracesof DMF from the raw product an additional dialysis in CHCl₃ wasperformed.

Conversion: quant.; yield: 86%; ¹HNMR (400 MHz, CDCl₃): δ=4.23-2.87(PG), 1.81 (PG-starter), 0.85 (PG-starter); ¹³CNMR (100 MHz, CDCl₃):δ=81.9-67.5 (PG), 60.5 (functionalised secondary PG-groups), 51.5(functionalised primary PG-groups); IR (KBr): v=2873, 2361, 2102 (N3),1457, 1273, 1122, 668 cm⁻¹.

3b) Synthesis of Polyglycerylamine 13

Polyglycerylazide 12 (16.50 g, 166.7 mmol N₃-groups) was dissolved inp.a. THF (150 ml) in a 500 ml one-necked flask. H₂O (10 ml) and PPh₃(43.67 g, 166.7 mmol, 1 eq.) were added and N₂ formation was observed.While stirring for 16 h, the amount of water in the reaction mixture wasincreased continuously by dropwise addition of H₂O (140 ml) via dropfunnel to avoid precipitation of the partially reduced product. Themixture was concentrated in vacuo to a smaller volume, CHCl₃ was addedand the phases were separated using a separation funnel. The aqueouslayer was extracted with CHCl₃ four times and then concentrated todryness to deliver a brown honey-like product 13, which was dialysed inMeOH.

Conversion: 82%; yield: 90%; ¹HNMR (300 MHz, CD₃OD): δ=4.01-3.21 (PG),3.31-2.40 (functionalised PG-groups); ¹³CNMR (75 MHz, CD₃OD):δ=83.0-65.5 (PG), 55.5-43.6 (functionalised PG-groups); IR (KBr):v=3354, 2874, 2362, 2338, 2103, 1576, 1473, 1338, 1104, 820, 668 cm⁻¹.

EXAMPLE 4 Gene Transfection In Vitro

Polyglyceryl pentaethylenehexamine carbamate 7 (in the followingreferred to as WF 33 or PG-(C₂H₄NH)₅) and polyglyceryl amine 13 (in thefollowing referred to as SX 118 or PG-NH₂) were found to be particularlyefficient for gene transfection as will be described in detail in thefollowing. The term “gene transfection” is to be understood as“transfection with a polynucleotide or oligonucleotide”.

The polynucleotides or oligonucleotides used in these experiments areable to bind to mRNA of the examined genes and to act via RNAinterference as gene silencer. In this context, “expression of proteins”is to be understood as gene transcription and subsequent translation ofmRNA into proteins. Thus, if a protein expression is diminished or atleast partially silenced, the production of said protein is reduced ascompared to usual levels at the step of gene transcription or mRNAtranslation, in particular at the step of mRNA translation by siRNA genesilencing.

Transfection experiments were done in the HeLaS3 cell line withdifferent proteins (Lamin, CDC2, MAPK2). For the sake of simplicity, theproteins will be partially named hereinafter like their encoding genes,although other names are also common for the examined proteins, like,e.g., CDK1 or CDC28A for the protein encoded by CDC2 (cell divisioncycle 2).

The results of the gene transfection efficiencies of PG-(C₂H₄NH)₅ (WF33)and PG-NH₂ (SX118) are shown in FIGS. 4 to 6. The results were comparedto the control transfection reagent HiPerFect (Qiagen) which is the invitro benchmark transfection reagent. PG-(C₂H₄NH)₅ and PG-NH₂ were foundto be highly efficient and thus very well suited for transfection ofHeLaS3 cells.

In a siRNA gene silencing experiment against expression of Lamin proteinin HeLaS3 cells, the results of which experiment are shown in FIG. 4, aconstant amount of 100 nM (nmol/l) siRNA was treated with 3 μl or 6 μlof different transfection reagents (siRNA carriers).

In FIG. 4, the first column shows the expression of the protein withoutany treatment (control experiment). The Lamin expression reached in thiscontrol experiment was set to 100%.

The second two columns of FIG. 4 show the expression of the proteinafter treatment with HiPerFect as transfection reagent. The next twocolumns show the expression of Lamin after treatment with PG-(C₂H₄NH)₅as transfection reagent and the last two columns show the expression ofLamin after treatment with PG-NH₂ as transfection agent. In each case,unfilled columns represent results obtained with 3 μl transfectionreagent and diagonally hatched columns represent results obtained with 6μl transfection reagent. All transfection reagents were equallyconcentrated.

As can be seen from FIG. 4, at a concentration of 100 nM siRNA,treatment with siRNA loaded transfection agents PG-(C₂H₄NH)₅ and PG-NH₂resulted in efficient silencing of Lamin expression (14 to 21% of usualexpression) which was comparable to that of silencing achieved withHiPerFect (12 to 15% of usual expression).

FIG. 5 shows the results of a siRNA gene silencing experiment againstexpression of CDC2 protein in HeLaS3 cells. The same siRNA carriers ortransfection reagents as in the experiment against Lamin expression wereused. However, 6 μl of each transfection reagent were used in allexperiments. On the other hand, different concentrations (25, 50 and 100nM) of siRNA were tested.

In FIG. 5, the first column shows the expression of the protein withoutany treatment (control experiment). The CDC2 expression reached in thiscontrol experiment was set to 100%.

The second three columns of FIG. 5 show the expression of the proteinafter treatment with HiPerFect as transfection reagent. The next threecolumns show the expression of CDC2 after treatment with PG-(C₂H₄NH)₅ astransfection reagent and the last three columns show the expression ofCDC2 after treatment with PG-NH₂ as transfection agent. In each case,unfilled columns represent results obtained with 100 nM siRNA,diagonally hatched columns represent results obtained with 50 nM siRNAand horizontally hatched columns represent results obtained with 25 nMsiRNA.

Whereas CDC2 expression could be silenced with HiPerFect as transfectionagent at all three siRNA concentrations chosen, silencing of CDC2expression was more efficient at a siRNA concentration of 100 nM ascompared to the other concentrations chosen in case of PG-(C₂H₄NH)₅ andPG-NH₂ as transfection reagents. Nonetheless, efficient silencing waspossible with PG-(C₂H₄NH)₅ and PG-NH₂, though the extent of silencingwas lower than that found with respect to Lamin expression (cf. FIG. 4).

FIG. 6 shows the results of a siRNA gene silencing experiment againstexpression of MAPK2 (Mitogen activated kinase 2) in HeLaS3 cells. Thesame siRNA carriers or transfection reagents as in the experimentagainst Lamin or CDC2 expression were used. Like in the experimentagainst CDC2 expression, 6 μl of each transfection reagent were used inall experiments and different concentrations (25, 50 and 100 nM) ofsiRNA were tested.

In FIG. 6, the first column shows the expression of the protein withoutany treatment (control experiment). The MAPK2 expression reached in thiscontrol experiment was set to 100%.

The second three columns of FIG. 6 show the expression of the proteinafter treatment with HiPerFect as transfection reagent. The next threecolumns show the expression of MAPK2 after treatment with PG-(C₂H₄NH)₅as transfection reagent and the last three columns show the expressionof MAPK2 after treatment with PG-NH₂ as transfection agent. In eachcase, unfilled columns represent results obtained with 100 nM siRNA,diagonally hatched columns represent results obtained with 50 nM siRNAand horizontally hatched columns represent results obtained with 25 nMsiRNA.

MAPK2 expression could be silenced with HiPerFect as transfection agentat all three siRNA concentrations chosen. Silencing of MAPK2 expressionwas also particularly efficient at a siRNA concentration of 100 nM anduse of PG-NH₂ as transfection reagent (20% of the usual expression).With PG-(C₂H₄NH)₅ as transfection reagent, 54% expression was the lowestvalue achieved at a siRNA concentration of 100 nM. At siRNAconcentrations of 50 or 25 nM, silencing with PG-(C₂H₄NH)₅ astransfection reagent was not that efficient as compared to HiPerFect astransfection reagent. Also, a concentration of 25 nM siRNA seemed to betoo low for efficient silencing of MAPK2 expression when PG-NH₂ was usedas transfection agent (93% of the usual expression was still observed inthis case).

In another experiment, silencing of luciferase gene by usingPG-(C₂H₄NH)₅ and PG-NH₂ as transfection reagent (or nanocarrier forrespective siRNA) was examined. For this experiment, siRNA of luciferasegene was entrapped into PG-(C₂H₄NH)₅ and PG-NH₂ and transfected into U87luciferase-infected cells (U87-Luc). Cells were harvested 48 h later andlysed. Luciferase activity was measured using a luminometer.

In FIG. 7, luciferase activity (in percent of a negative control withoutany treatment) is plotted versus different concentrations (on alogarithmic scale) of PG-(C₂H₄NH)₅ and PG-NH₂ as nanocarriers comprising100 nM luciferase siRNA. Thus, the silencing efficiency of PG-(C₂H₄NH)₅and PG-NH₂ loaded with luciferase siRNA can be seen from FIG. 7.

EXAMPLE 5 Cytotoxicity Profiling of PG-(C₂H₄NH)₅ and PG-NH₂

Since PG-(C₂H₄NH)₅ and PG-NH₂ were found to be efficient fortransfection into HeLaS3 Cell lines and also showed higher silencing ofluciferase gene, they were tested for their cytotoxicity behaviour. Inboth cases almost no cytotoxicity was observed in case of administering3 μl transfection agents (cf. FIG. 8). However, the toxicity shows anincrement with increasing dose of transfection agents as can be seenfrom experiments using 6 μl of the respective transfection agents (cf.FIG. 9).

In FIGS. 8 and 9, in each case the results of two independentmeasurements are depicted. Results of the first experiments arerepresented by unfilled columns and results of the second experimentsare represented by diagonally hatched columns.

Results obtained with respect to “no treatment” are a negative controlrepresenting the normal rate of cytotoxicity observed in a cell culturedue to usual biological processes, such as apoptosis.

“High control” refers to a positive control. 24 h after transfection ofthe cells, 40 μl of a 10% (w/v) Triton 100 solution were added to themedium of non-transfected cells. After incubation at 37° C. for 1 h, thesupernatant was removed and centrifuged. 100 μl of the resultingsupernatant was then subjected to the cytotoxicity assay (for details,see below).

Cytotoxicity of PG-(C₂H₄NH)₅ and PG-NH₂ was also tested for SK—N—SHneuroblastoma, Kelly neuroblastoma and U87 glioblastoma cells. The celllines were challenged with respective nanocarriers at serialconcentrations. Cells were counted 72 h later, by XTT (tetrazoliumhydroxide) reagent. The use of tetrazolium salts, such as MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), is basedon the fact that living cells reduce tetrazolium salts into coloredformazan compounds.

The biochemical procedure is based on the activity of mitochondriaenzymes which are inactivated shortly after cell death. This method wasfound to be very efficient in assessing the viability of cells. Acolorimetric method based on the tetrazolium salt, XTT, was developed in1988. Whilst the use of MTT produced a non-soluble formazan compoundwhich necessitated dissolving the dye in order to measure it, the use ofXTT produces a soluble dye and simplifies the procedure of measuringproliferation, and is, therefore, an excellent solution to thequantification of cells and their viability without using radioactiveisotopes. This kit was developed to assay cell proliferation in reactionto different growth factors, cytokines, nutrient components and drugs.

Results are presented as % of the control (proliferation without anytreatment) in FIGS. 10A to 10C.

FIG. 10A shows the cytotoxicity of PG-NH₂ and PG-(C₂H₄NH)₅ on SK—N—SHneuroblastoma cells, FIG. 10B shows the cytotoxicity of PG-NH₂ andPG-(C₂H₄NH)₅ on Kelly neuroblastoma cells and FIG. 10C shows thecytotoxicity of PG-NH₂ and PG-(C₂H₄NH)₅ on U87 human glioblastoma cells.In each case, different concentrations of the dendritic nanocarriersPG-NH₂ and PG-(C₂H₄NH)₅ were tested. As explained above, living cellswere counted by XTT assay 72 h after adding nanocarriers. Results arepresented as % of the control (proliferation without any treatment).

Both PG-(C₂H₄NH)₅ and PG-NH₂ were not cytotoxic in a concentration of upto 10 μg/ml on the three tested cell lines. In this context,cytotoxicity of the nanocarriers is assumed to be given if theconcentration needed for gene silencing is significantly higher thantheir IC₅₀ value.

Table 1 shows the silencing efficiency versus toxicity of PG-(C₂H₄NH)₅and PG-NH₂, on U87-Luc human glioblastoma cells obtained from thatexperiments, the results of which are depicted in FIG. 7 (forcytotoxicity experiments, see also example 5). These architecturesdemonstrated the best silencing efficiency and safety in U87-Luc cellsand therefore were selected for examining in vivo cytotoxicity and genesilencing efficacy studies.

TABLE 1 Silencing efficiency of luciferase gene and cytotoxicity onU87-Luc human glioblastoma cells of PG—(C₂H₄NH)₅ and PG—NH₂. Nanocarrier50% silencing 70% silencing IC₅₀ PG—(C₂H₄NH)₅ 17 μg/ml 30 μg/ml 55 μg/mlPG—NH₂  8 μg/ml 12 μg/ml 30 μg/ml

As can be seen from Table 1, the concentration at which 50% of theexamined cells were inhibited by PG-(C₂H₄NH)₅ or PG-NH₂ (the IC₅₀ value)was 55 and 30 μg/ml respectively. At almost half of this concentration,70% silencing (silencing being defined as “1—expression”) was achievedwhen using PG-(C₂H₄NH)₅ as nanocarrier for siRNA of luciferase gene.With PG-NH₂ as according nanocarrier, 70% silencing was already achievedat an even lower concentration (12 μg/ml) with respect to U87-Luc cells.

In order to further evaluate the biocompatibility of the nanocarrierswith red blood cells, a Red Blood Cells Lysis assay (Duncan, R.,Ferruti, P., Sgouras, D., Tuboku-Metzger, A., Ranucci, E., and Bignotti,F. J Drug Target, 2:341-347, 1994) was performed.

Fresh blood was obtained from male wistar rats (˜250 g) by cardiacpuncture and collected in heparinized tubes. The erythrocytes were thenwashed 3-4 times with pre-chilled PBS, to finally make a red blood cell(RBC) solution of 2% w/w in PBS. Double strength solutions of thecarriers tested were prepared and 100 μl plated into a 96 well plate.Negative controls were PBS (blank) and Dextran (MW ˜70 000): positivecontrols were 1% w/v solution of Triton X100 (100% lysis) andpoly(ethylene imine) (PEI). 100 μl of the 2% w/w RBC stock solution wasadded to each well and incubated for 1 hour at 37° C. Followingcentrifugation at 1000*g for 10 minutes at RT, the supernatant was drawnoff and its absorbance measured at 550 nm using a microplate reader(Genios, TECAN). The results were then expressed as % of haemoglobinreleased relative to the positive control (Triton X100).

The results clearly show that at concentrations up to 1 mg/ml thedendrimers were not haemolytic in vitro (FIG. 10D). In detail, FIG. 10Dshows the results of the Red Blood Cells Lysis assay. A rat red bloodcells solution (2% w/v in PBS) was added to a previously prepared96-wells plate containing the tested compounds at serial concentrations(▪ PG-NH₂, ▴ PG-(C₂H₄NH)₅, □ Dextran, x PEI). Following incubation at37° C. for 1 hour and centrifugation, haemoglobin release was measuredspectrophotometrically (OD₅₅₀), using PBS as a blank. Results arepresented as % of the released hemoglobin produced by TritonX-100±standard deviation.

Summarizing, dendritic nanocarriers are non-toxic at the concentrationsrequired for gene silencing in vitro.

EXAMPLE 6 In Vivo Silencing the Luciferase Gene by siRNA

In order to demonstrate the potential of the novel siRNA-nanocarriers incancer therapy, preliminary in vivo experiments were performed.

All animal procedures were performed in compliance with Tel AvivUniversity, Sackler School of Medicine guidelines and protocols approvedby the Institutional Animal Care and Use Committee.

On the one hand, human glioblastoma U87-Luciferase cells (10⁶ cells in100 μl phosphate buffered saline (PBS)) were injected subcutaneouslyinto SCID (Severe Combined Immunodeficiency) mice (male, 6 to 8 weeksold). Once the tumors grew to a volume of about 100 mm³, 7 or 21 nmol(3.6 or 10 mg/kg) luciferase siRNA mixed together with 56 or 170 nmol(20 or 60 mg/kg) SX 118, respectively, were injected intratumorally(t=0). The tumor volume was measured daily with a caliper. The silencingefficiency of the luciferase siRNA encapsulated in the SX118nanoparticle or nanocarrier was followed up by non-invasive intravitalbioluminescence imaging system (Biospace Photon Imager) followingluciferin injection intraperitoneally (50 mg/kg).

The luciferase activity of mice treated with different amounts ofluciferase siRNA entrapped in SX118 was followed up by non-invasiveintravital bioluminescence imaging for 3 days following treatment. Mice1 and 2 were injected with 3.6 mg/kg luciferase siRNA entrapped in 20mg/kg SX118; and mouse 3 was injected with 10 mg/kg luciferase siRNAentrapped in 60 mg/kg SX118.

FIG. 11A depicts luciferase activity within the generated tumor atdifferent measurement times as bioluminescence images.

FIG. 12A represents results as % of the initial luciferase activity(before any treatment), normalized to tumor volume. Complete genesilencing was accomplished in vivo within 24 hours following treatmentwith the high dose luciferase siRNA-SX118 complex (mouse 3), while 80%silencing was achieved following treatment with the low dose complex(mice 1 and 2) as measured by photon flux bioluminescence.

On the other hand, SCID mice male aged 6-8 weeks (Harlan LaboratoriesIsrael LTD) were anesthesized by ketamine (150 mg/kg) and xylazine (12mg/kg) and inoculated subcutaneously (s.c.) with 1×10⁶ U87-Luciferasecells.

Tumor progression was monitored by caliper measurement(width×length²×0.52). Mice were imaged by bioluminescence imaging system(Biospace Photon Imager), following an intraperitoneal (i.p.) injectionof luciferin (50 mg/kg). Photons were collected for a period of 15minutes, images were obtained by Photonvision+ software (Biospace) andresults analysis was performed by Molecular Vision software (Biospace).

Mice bearing 70 mm³ tumors were injected intratumorally with luciferasesiRNA-PGNH₂ and luciferase siRNA-PG(C₂H₄NH)₅ complexes at two differentconcentrations, siRNA alone or saline, as detailed in the table 2.

TABLE 2 Experimental Details of intratumoral injection. PG—NH₂PG(C₂H₄NH)₅ siRNA + siRNA + siRNA alone Saline siRNA 2.5, 5   2.5, 5  2.5, 5 — (mg/kg) Vehicle 10, 20 15, 30 — — (mg/kg)

Human glioblastoma U87-Luciferase cells were inoculated subcutaneouslyinto SCID mice. Once tumors developed, anti-luciferase-siRNA complexedwith PG-NH₂ or PG-(C₂H₄NH)₅ was injected intratumorally. Mice wereadministered with two injections of the different treatments, on day 0and day 4. Animals were monitored 3 times a week for general health,body weight, tumor volume and luciferase activity following treatment.

A significant gene silencing (32% and 15%) was accomplished in vivowithin 24 hours following treatment with luciferase siRNA complexed withPG-NH₂ (2.5 (low dose) and 5 mg/kg (high dose) siRNA complexed with 10and 20 mg/kg PG-NH₂ respectively), as measured by photon fluxbioluminescence (FIGS. 11B and 12B).

In detail, in FIGS. 12B and 13B black squares denote a high dose, andgrey squares denote a low dose of anti-luciferase siRNA complexed withPG-NH₂. Further, black triangles denote a high dose, and grey trianglesdenote a low dose of anti-luciferase siRNA complexed with PG-(C₂H₄NH)₅.As a control, saline was injected to control mice (indicated by whitesquares). Mice received 2 injections, on day 0 and on day 4.

Low levels of luciferase activity were maintained for 3-4 days after asingle dose of siRNA-nanocarrier, and even for 3 days more, following asecond dose of the treatment on day 4. Similar results were obtainedwithin 1-3 days following treatment with luciferase siRNA entrapped inthe nanocarrier PG-(C₂H₄NH)₅. However, in this case, the silencingeffect was not prolonged by a second dose at day 5 (FIG. 12, black andgrey triangles). No significant weight loss occurred following twoconsecutive intratumoral injections of all complexes of dendriticvehicles and siRNA at two different doses to SCID mice (FIG. 13B).

Summarizing, intratumoral injection of PG-NH₂-siRNA andPG-(C₂H₄NH)₅-siRNA complexes leads to in vivo gene silencing.

EXAMPLE 7 Biocompatibility of Intravenously Administered PG-NH₂ In Vivo

Further, preliminary in vivo biocompatibility tests for intravenousadministration of PG-NH₂ (FIG. 13) were performed.

Firstly, mice were injected intravenously with increasing doses of SX118 (0.0125, 0.125 and 12.5 mg/kg). Body weight, general physicalcondition and behaviour of the mice were monitored during 15 daysfollowing treatment. No significant change in the physical state orbehaviour of the animals was observed during this period.

The percent of change in body weight 4, 11 and 15 days followingtreatment is plotted in FIG. 13A. Whereas at a high concentration of SX118 injected, virtually no significant change in body weight can be seenafter 15 days, an increase in body weight could be observed after 15days in case of animals treated with low and medium doses of SX 118.Generally, an increase in body weight can be seen as an indication of agood physical state of an animal.

Secondly, PG-NH₂ (5 mg/kg (low dose) and 10 mg/kg (high dose)), withoutsiRNA, was administered by injection into the tail vein of 6-8 weeks oldmale FVB mice (a commonly used mice strain). Animals were monitoreddaily for general health and changes in body weight during 14 daysfollowing treatment.

In FIG. 13C the percent change in the body weight of the mice isplotted. Black squares denote a high dose, and grey squares denote a lowdose of PG-NH₂.

No significant change in the body weight of the mice was observed.General health and behaviour were also monitored and found to besuitable.

Summarizing, the synthetic protocols and biological experiments of theamine terminated PG herein disclosed reveal that such polymericscaffolds hold high possibilities to transfect siRNA into the cellularmatrix with appreciable biocompatibility. The polycationic architectureof these systems provides them with rapid cellular uptake and longernuclear residence time.

EXAMPLE 8 Determination of the Average Size by Dynamic Light Scattering(DLS)

The mean hydrodynamic diameter of the nanocarriers prepared according toexamples 1 to 3 was evaluated using a real time particle analyzer(NanoSight LM20™) containing a solid-state, single mode laser diode (<20mW, 655 nm) configured to launch a finely focused beam through a 500 μlsample chamber. PG-NH₂ and PG-(C₂H₄NH)₅ were dissolved in phosphatebuffered saline (PBS) to a final concentrations of 1 mg/ml. The sampleswere then injected into the chamber by syringe and allowed toequilibrate to unit temperature (23° C.) for 30 seconds. The particlesdynamics were visualized at 30 frames per second (fps) for 60 seconds at640×480 resolution by the coupled charge device (CCD) camera. The pathsthe particles took under Brownian motion over time were analyzed usingNanoparticle Tracking Analysis (NTA) software. The diffusion coefficientand hence sphere equivalent hydrodynamic diameter of each particle wasseparately determined and the particle size distribution profiles weregenerated. Each sample was measured three times in triplicates, and theresults represent the mean diameter.

The results can be seen from FIG. 14. In particular for PG-NH₂, twopronounced populations of differently sized particles can be observed.

EXAMPLE 9 Polyplex Formation Study Between the Dendritic Nanocarriersand siRNA

For this and the following experiments, Fluorescein isothiocyanate(FITC)-labeled or Cyanine-3 (Cy3)-labeled anti-luciferase siRNA (5′ GAUUAU GUC CGG UUA UGU AUU 3′) and non-targeting siRNA purchased fromDharmacon were used. It is possible that the siRNA will be labeled witha certain marker and the dendritic polymer with a different marker.

The nanocarriers described in this and the following examples wereprepared according to examples 1 to 3.

In order to establish the capability of the four tested dendriticnanocarriers to form a complex with siRNA, several amounts of dendrimerswere incubated with an equal amount of siRNA and the efficacy of thecomplexes formation was analyzed by gel electrophoresis. The optimalratio for the polyplex formation was studied by Electrophoretic MobilityShift Assay (EMSA) as previously described (Kumar, P., Wu, H., McBride,J. L., Jung, K. E., Kim, M. H., Davidson, B. L., Lee, S. K., Shankar,P., and Manjunath, N, Nature, 448: 39-43, 2007). Briefly, 100 pmol ofsiRNA was incubated with PG-NH₂, PG-(C₂H₄NH)₅, PEI-PAMAM (i.e.,poly(ethylene imine)-polyamidoamine) or PEI-Gluconolacton (i.e.poly(ethylene imine) gluconolacton) at 5:1, 2:1, 1:1, 1:2 and 1:5 molarratios of siRNA to carrier, for 15 minutes at room temperature. Mobilityof free or nanocarrier-complexed siRNA was then analyzed by agarose-gelelectrophoresis.

The results of an according electrophoresis mobility shift assay can beseen from FIG. 15. All four nanocarriers were able to bind siRNA andneutralize its negative charge in a dose-dependent manner, as shown byan electrophoretic mobility shift assay. No significant differencesbetween the ability of the four tested cationic carrier systems toencapsulate the siRNA were detected.

EXAMPLE 10 Intracellular Trafficking of siRNA-PG-NH₂ Complex by ConfocalMicroscopy and by ImageStream Multispectral Imaging Flow Cytometer

U87 human glioblastoma and human embryonic kidney 293T (HEK 293T) usedin this and the following experiments were obtained from the AmericanType Culture Collection (ATCC). Cells were cultured in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum(FBS), 100 μg/ml Penicillin, 100 U/ml Streptomycin, 12.5 U/ml Nystatinand 2 mM L-glutamine (Biological Industries, Israel). Cells were grownat 37° C. in 5% CO₂.

For establishment of luciferase-infected U87 human glioblastoma cellline, HEK 293T cells were co-transfected with pLBLuc and the compatiblepackaging plasmids (pMD.G.VSVG and pGag-pol.gpt). Forty eight hoursfollowing transfection, the pLB-Luc retroviral particles containingsupernatant were collected. U87 cells were infected with the retroviralparticles media, and 48 hours following the infection, luciferasepositive cells were selected by hygromycin resistance.

U87-Luc cells were plated at 2×10⁶ cells/6 cm culture plate inDulbecco's Modified Eagle Medium (DMEM), supplemented with 10% fetalbovine serum (FBS) for 24 hours. (FITC)-labeled Luciferase siRNA (100nM) was mixed with PG-NH₂ (20 μg/ml) in serum free medium, incubated for20 minutes at room temperature, and then added to the cells. Severaltime points following treatment with siRNA-PG-NH₂ complex (0, 2, 5 and24 hours) cells were harvested, resuspended in 100 μl PBS, and analyzedby ImageStream multispectral imaging flow cytometer (Amnis Corp.). Theplatform produces high resolution brightfield, and fluorescence imagesof live cells prepared in suspension at rates up to 100 cells persecond. The IDEAS™ analysis software quantifies over 200 morphometricand photometric parameters for each cell based on its imagery.

Following chemical characterization, the ability of Cy3-labeledanti-luciferase siRNA entrapped in PG-NH₂ to internalize into humanglioblastoma cells was evaluated. U87 cells were incubated with thesiRNA-PG-NH₂ 2, 5 and 24 hours following transfections. Cells werefixed, permeabilized, stained and analyzed by confocal microscopy.

In detail, U87-Luc cells were plated at 100,000 cells/well onto coverslides on 24-well culture plates in DMEM, supplemented with 10% fetalbovine serum (FBS) for 24 hours. Luciferase siRNA (100 nM) was mixedwith PG-NH₂ (20 μg/ml) in serum free medium, incubated for 20 minutes atroom temperature, and then added to the cells. U87 cells were incubatedwith siRNA-PG-NH₂ complex for 2, 5 and 24 hours or in the absence ofsiRNA, then washed several times with cold PBS, fixed with 4%paraformaldehyde (15 min, room temperature (RT)) and washed with PBSagain. Actin filaments were labeled using phalloidin-FITC conjugate (25μg/ml, 40 minutes at RT), nuclei were labeled using Hoechst staining (1μg/ml, minutes at RT) and cover glasses were then mounted with Antifade®mounting media. Cellular uptake and internalization were monitoredutilizing a Leica TCS SP5 confocal imaging system. All images were takenusing a multi-track channel acquisition to prevent emission cross-talkbetween fluorescent dyes.

Thus, U87 cells were transfected with Cy3-labeled anti-luciferase siRNAencapsulated in PG-NH₂. The cells were fixed at the indicated timepoints following transfection, and analyzed by confocal microscopy(Leica TCS SP5). Following 2 hours incubation, siRNA was alreadydetected inside the cells, accumulated mostly in the cytoplasm, asobserved in the single plane image (FIG. 16A, 2 hours). FIG. 16A depictssingle XY plane imaging of the siRNA (red) cytoplasmic accumulation, atthe indicated times following transfection. Cells were stained withFITC-phalloidin (green) for actin filaments and DAPI (blue) for nuclei.The amount of siRNA accumulated inside the cells was significantlyhigher 3 hours later, and even more, the day after the transfection(FIG. 16A, 5 hours and 24 hours respectively). To evaluate the siRNAcellular localization and eliminate optical artefacts, an X, Z slice wascaptured and analyzed (FIG. 16C, lower panel). FIG. 16C shows XZ imageslice (lower panel) of a cell fixed 5 hours following siRNA-PG-NH₂transfection (upper panel) revealed similar siRNA, actin filaments andnuclei focal plane localization. Scale bars represent 25 μm and 10 μmfor the XZ image slice. siRNA was located at the same focal plane asactin (stained by the FITC-labeled phalloidin), confirming itsintracellular uptake.

Further examination of the cellular internalization of siRNAencapsulated in PG-NH₂ was performed by ImageStream multispectralimaging flow cytometer. Live cells were monitored different timesfollowing transfection, using FITC-labeled siRNA and Cy3-labeledphalloidin (red) for actin filaments, and siRNA intracellular uptake wasmonitored in live cells, at the indicated times following transfection(FIGS. 16E, 16F and 16G). PG-NH₂ was capable of delivering the siRNAinto U87 cells as demonstrated by increasing levels of fluorescencemeasured inside the cells 2, 5 and 24 hours following transfection.

Summarizing, dendritic nanocarriers entrap siRNA and deliver it intohuman glioblastoma cells in vitro.

EXAMPLE 11 Further Dendritic Nanocarriers

FIG. 17 shows the chemical structures of the natural polyaminesputrescine, spermidine, spermine and its analogues diethylenetriamine,bis(3-aminopropylamine) and pentaethylenhexamine. These polyamines canalso be used to modify polyglycerol in order to produce nanocarriersaccording to the invention. The synthesis can be generally done asdescribed above, particularly with respect to examples 1 to 3. However,alternative reaction methods are possible.

In FIG. 18A and FIG. 18B such alternative methods are schematicallydepicted. Thus, FIG. 18A shows a glycol oxidation of polyglycerol andreductive amination (reaction a) and a glycol oxidation and formation ofa Schiff base (reaction b), both ways leading to nanocarriers accordingto the invention. “r.t.” stands for room temperature.

FIG. 18B shows the reaction scheme of a synthesis of pH cleavablesystems through Mitsunobu reaction.

1-18. (canceled)
 19. A compound suited as entity carrier, having thegeneral formula (I)

with PG denoting a linear or branched polyglycerol core, X being

and being covalently bound to a carbon atom of the polyglycerol core,wherein the polyglycerol core carries a plurality of groups of the typeX, R¹ being H, linear or branched C₁-C₁₀-Alkyl, which may be substitutedand/or interrupted by one or more oxygen, sulphur and/or nitrogen atoms,or R³, R^(2′) being linear or branched C₁-C₁₀-Alkyl, which may besubstituted and/or interrupted by one or more oxygen, sulphur and/ornitrogen atoms, or R³, R³ being

R⁴ being H or C₁-C₄-Alkyl, which may be substituted and/or interruptedby one or more oxygen, sulphur and/or nitrogen atoms, and n being 1 to100, wherein R¹ and R^(2′) cannot simultaneously be an ethyl residue.20. A compound according to claim 19, characterized in that at least50%, of the free hydroxyl groups of the polyglycerol core aresubstituted by groups of the type X.
 21. A compound according to claim20, characterized in that all of the free hydroxyl groups of thepolyglycerol core are substituted by groups of the type X.
 22. Acompound according to claim 19, characterized in that n is 1 to 10,particularly
 5. 23. A compound according to claim 19, characterized inthat R⁴ is H.
 24. A compound according to claim 19, characterized inthat R¹ is a methyl residue and R^(2′) is an N-dimethyl ethyl amineresidue.
 25. A compound according to claim 19, characterized in that atleast one of R¹ and R² is selected from the group consisting of H andsaid R³.
 26. A compound according to claim 19, characterized in that thecompound bears at least one functional group of the general formula

wherein residues R³ and R⁴ have the above-defined meanings. 27.Nanocarrier system comprising a compound according to claim 19 and atleast one entity to be carried by and bound to said compound in acovalent, ionic or complexed manner, wherein said entity is chosen fromthe group comprising nucleotides, nucleosides, linear or circular singleor double stranded oligonucleotides, oligomeric molecules comprising atleast one nucleoside, small pharmacologically active molecules having amolecular mass of not more than 800 g/mol, amino acids, peptides, andmetal ions.
 28. A nanocarrier system according to claim 27,characterized in that at least 50%, of the free hydroxyl groups of thepolyglycerol core are substituted by groups of the type X.
 29. Ananocarrier system according to claim 28, characterized in that all ofthe free hydroxyl groups of the polyglycerol core are substituted bygroups of the type X.
 30. A nanocarrier system according to claim 27,characterized in that R¹ is a methyl residue and R^(2′) is an N-dimethylethyl amine residue.
 31. A nanocarrier system according to claim 27,characterized in that at least one of R¹ and R² is selected from thegroup consisting of H and said R³.
 32. A nanocarrier system according toclaim 27, characterized in that the compound bears at least onefunctional group of the general formula

wherein residues R³ and R⁴ have the above-defined meanings. 33.Nanocarrier system, comprising at least one nanocarrier being a compoundhaving a structure according to formula (I),

wherein PG denotes a linear or branched polyglycerol core, and X being

and being covalently bound to a carbon atom of the polyglycerol core,wherein the polyglycerol core carries a plurality of groups of the typeX, R¹ being H, linear or branched C₁-C₁₀-Alkyl, which may be substitutedand/or interrupted by one or more oxygen, sulphur and/or nitrogen atoms,or R³, R² being H, linear or branched C₁-C₁₀-Alkyl, which may besubstituted and/or interrupted by one or more oxygen, sulphur and/ornitrogen atoms, or R³, R³ being

and n being 1 to 100, at least one entity to be carried by and bound tosaid nanocarrier in a covalent, ionic or complexed manner, wherein saidentity is chosen from the group comprising nucleotides, nucleosides,linear or circular single or double stranded oligonucleotides,oligomeric molecules comprising at least one nucleoside, smallpharmacologically active molecules having a molecular mass of not morethan 800 g/mol, amino acids, peptides, and metal ions, with theprovision that the entity does not comprise a double stranded circularand covalently closed nucleic acid having a length of more than 1000bases or base pairs, preferably of more than 500 bases or base pairs.34. A nanocarrier system according to claim 33, characterized in thatthe entity is a ribonucleic oligonucleotide.
 35. A nanocarrier systemaccording to claim 34, characterized in that the ribonucleicoligonucleotide has a length of 10 to 40 bases.
 36. A nanocarrier systemaccording to claim 33, characterized in that at least 50% of the freehydroxyl groups of the polyglycerol core are substituted by groups ofthe type X.
 37. A nanocarrier system according to claim 33,characterized in that at least one of R¹ and R² is selected from thegroup consisting of H and said R³.
 38. A nanocarrier system according toclaim 33, characterized in that the compound bears at least onefunctional group of the general formula

wherein residues R³ and R⁴ have the above-defined meanings.
 39. Apharmaceutical composition comprising the compound of claim 19, thecomposition being for use as a carrier for an entity, wherein the entityis chosen from the group comprising nucleotides, nucleosides, linear orcircular single or double stranded oligonucleotides, oligomericmolecules comprising at least one nucleoside, small pharmacologicallyactive molecules having a molecular mass of not more than 800 g/mol,amino acids, peptides, and metal ions, with the provision that theentity does not comprise a double stranded circular and covalentlyclosed nucleic acid having a length of more than 1000 bases or basepairs, preferably of more than 500 bases or base pairs.
 40. Apharmaceutical composition according to claim 39, wherein the compoundis characterized in that at least 50% of the free hydroxyl groups of thepolyglycerol core are substituted by groups of the type X.
 41. Apharmaceutical composition according to claim 39, wherein the compoundis characterized in that at least one of R¹ and R² is selected from thegroup consisting of H and said R³.
 42. A pharmaceutical compositionaccording to claim 39, wherein the compound is characterized in that thecompound bears at least one functional group of the general formula

wherein residues R³ and R⁴ have the above-defined meanings.
 43. A methodof transporting an entity into at least one human or animal cell, themethod comprising contacting the cell with a nanocarrier systemaccording to claim
 27. 44. A method of transporting an entity into atleast one human or animal cell, the method comprising contacting thecell with a nanocarrier system according to claim 33, with the provisionthat the entity does not comprise a double stranded circular andcovalently closed nucleic acid having a length of more than 1000 basesor base pairs, preferably of more than 500 bases or base pairs.
 45. Kitfor performing transfection reactions, comprising a compound accordingto claim 19 in any suited formulation.
 46. Kit for performingtransfection reactions, comprising a nanocarrier system according toclaim 27 in any suited formulation.
 47. Kit for performing transfectionreactions, comprising a nanocarrier system according to claim 33 in anysuited formulation.
 48. A method of silencing a gene in vitro, ex vivo,in situ or in vivo, characterized by applying a nanocarrier systemaccording to claim 33 into at least one human or animal cell, whereinthe entity is a ribonucleic oligonucleotide.
 49. A method according toclaim 48, characterized in that the gene to be silenced is a tumorrelated gene.
 50. A method for the treatment of cancer, characterized byadministering a nanocarrier system according to claim 27 to at least onehuman or animal being.
 51. A method according to claim 50, characterizedin that the treatment of cancer is performed as combination treatment bya combined administering of a known anti-cancer or anti-angiogenic drug.52. A method for the treatment of cancer, characterized by administeringa nanocarrier system according to claim 33 to at least one human oranimal being.
 53. A method according to claim 52, characterized in thatthe treatment of cancer is performed as combination treatment by acombined administering of a known anti-cancer or anti-angiogenic drug.54. A method for the treatment of cancer, characterized by administeringto at least one animal or human being a nanocarrier system comprising:at least one nanocarrier being a compound having a structure accordingto formula (I),

wherein PG denotes a linear or branched polyglycerol core, and X being

and being covalently bound to a carbon atom of the polyglycerol core,wherein the polyglycerol core carries a plurality of groups of the typeX, R¹ being H, linear or branched C₁-C₁₀-Alkyl, which may be substitutedand/or interrupted by one or more oxygen, sulphur and/or nitrogen atoms,or R³, R² being H, linear or branched C₁-C₁₀-Alkyl, which may besubstituted and/or interrupted by one or more oxygen, sulphur and/ornitrogen atoms, or R³, R³ being

and n being 1 to 100, at least one entity to be carried by and bound tosaid nanocarrier in a covalent, ionic or complexed manner, wherein saidentity is chosen from the group comprising nucleotides, nucleosides,linear or circular single or double stranded oligonucleotides,oligomeric molecules comprising at least one nucleoside, smallpharmacologically active molecules having a molecular mass of not morethan 800 g/mol, amino acids, peptides, and metal ions.
 55. A methodaccording to claim 54, characterized in that the treatment of cancer isperformed as combination treatment by a combined administering of aknown anti-cancer or anti-angiogenic drug.